1. Technical Field
The invention relates to polypeptides which contain phosphorylated amino acid residues, pharmaceutical compositions which contain such polypeptides and the use of such pharmaceutical compositions for the prevention or treatment of conditions associated with bone loss or weakness.
2. Background Art
It is well-documented that disorders of bone and calcium metabolism cause numerous significant health problems on world-wide basis. For example, in the United States alone, diseases such as osteoporosis, renal osteodystrophy and Paget's Disease afflict over 2 million, 250 thousand, and 50 thousand patients, respectively, and the incidence appears to be increasing.
The majority of bone diseases are characterized by loss of bone minerals (particularly calcium), weakening of bones and consequently, an increase of the frequency and severity of bone fractures. In the elderly population, this has significant social ramifications as well, as many of those with bone fractures have difficultly with mobility, which often leads to the deterioration of other mental and physical functions, resulting in dementia, muscular weakness and/or fatigue. In addition, morbidity and pain are significantly increased by thrombotic events, such as pulmonary embolism which occur as a result of hip or pelvic fractures.
Bone is composed primarily of matrix proteins and calcium salts. Bone growth involves not only an increase in bone size, but an increase in the amount of such components as well. Such material growth of bone provides systemic and local mechanical strength of the skeleton. In the case of bone loss, a significant decline in the contents of these components, rather than in the size of the bone, is more frequently observed. This results in loss of mechanical strength and fractures which occur more frequently and are more severe.
As indicated above, one major function of bone is to provide mechanical strength. In other words, the skeleton needs to be strong enough to support the entire body weight and any additional mechanical burden. Therefore, bone size (mass) and strength must always correlate with whole body weight. Bone growth parallels that of the entire body, with respect to formation and calcium deposition. In humans, the maximum bone mass occurs around the age of 35 and is referred to as "peak bone mass." At peak bone mass, the amounts of matrix protein and calcium are also at their highest, and as a result, mechanical strength is greatest.
It is widely accepted that bone mineral content and density are correlated with the mechanical strength of the bone. After age 35, bone mass and mineral content and accordingly, mechanical strength, begin declining gradually. Consequently, when mechanical strength declines to a certain level, the individual is at greater risk of bone fracture. This natural occurrence is called osteoporosis if severe enough to be pathogenic.
The speed at which bone loss occurs differs among individuals, and especially with respect to gender. In females, the speed of bone loss accelerates immediately after menopause (See FIG. 1) because of a significant decline in available estrogen, a hormone which plays a critical role in maintaining healthy bone metabolism. Postmenopausal osteoporosis constitutes an important clinical problem because it afflicts significant numbers of women. Notably, the ratio of female to male osteoporosis patients is 3:1.
Another important function of bone is storage of calcium, an important cation in most biological functions. In mammals, calcium concentration in bodily fluids (such as blood) is kept within a range of 8.5 to 11 mg/dl (2.1 to 2.8 mM). This soluble calcium level is critical in maintaining normal biological functions. Excessive calcium stored in the bone is available for use as needed to maintain adequate levels in bodily fluids. When calcium levels become too low, the body first tries to absorb dietary calcium from the duodenum and intestine, and then if levels are still insufficient, calcium is obtained by dissolving the bone.
Absorption of dietary calcium is stimulated by Vitamin D, synthesized primarily in the kidney. In patients with renal failure who have insufficient intake of dietary calcium, the body must use calcium stored in the bone. This usually causes rapid bone loss and is called renal osteodystrophy (secondary osteoporosis caused by renal failure). Most patients with renal failure are afflicted with this condition regardless of whether they are on dialysis.
Metabolically, bone is a highly active organ with bone degradation (resorption) and reformation (remodeling) occurring continuously. Resorption is facilitated by osteoclasts which are differentiated from monocyte/macrophage lineage cells. Osteoclasts adhere to the surface of bone and degrade bone tissue by secreting acids and enzymes. Osteoblasts facilitate bone remodeling by adhering to degraded bone tissue and secreting bone matrix proteins, which differentiate into bone cells (osteocytes), and become a part of bone tissue. Bone remodeling occurs continuously throughout life.
Numerous experimental approaches have been attempted to either accelerate bone formation or diminish bone resorption. For example, factors such as BMPs (bone morphogenetic proteins), TGF-beta (transforming growth factor), IGF (insulin-like growth factor) are thought to have a significant role in bone formation. However, these factors have not been developed as therapeutic agents for systemic bone diseases. Furthermore, they are not suitable for therapeutic use because they are not available in oral form and cannot be selectively delivered to bone tissue. The fact that the processes of bone formation and resorption are so closely connected makes it extremely difficult to increase or decrease either process.
Type I collagen accounts for the majority of bone matrix proteins and comprises almost 90% of decalcified bone. Type I collagen is composed of three very large (MW&gt;100,000) protein chains which are mutually cross-linked by hydroxyproline (Hyp) and hydroxylysine (Hyl) residues. Other bone matrix proteins include osteocalcin, Matrix Gla Protein (MGP), Osteonectin, Bone Sialoprotein (BSP), Osteopontin, and Proteoglycans (PG-I and PG-II). These matrix proteins consist of approximately 10% of decalcified bone. Functions of these minor matrix proteins are not completely understood, however, as most of them are acidic proteins, it has been theorized that they may have a role in bone mineralization and/or demineralization.
Osteocalcin (MW=5,930) is composed of 49 amino acid residues which include three Gla (gamma-carboxyl glutamic acid) residues. This protein comprises 1 to 2% of total bone proteins. It is produced by osteoblast which is stimulated by Vitamin D3. It is an acidic protein and found in demineralization fluid. The function of this protein is thought to be to suppress excessive mineralization (Mikuni-Takagaki et al., Journal of Bone and Mineral Research (1995) 10(2):231-242).
Matrix Gla Protein is composed of 79 amino acids including 5 Gla residues. This rotein is usually found in demineralized matrix and believed to have a certain function in he initiation of bone formation.
Osteonectin is a glycoprotein (MW=30,000) which comprises about 20 to 25% of non-collagen matrix proteins in the bone. It has two N-glycosylation sites, and binds Type I collagen and hydroxyapatite. It contains a high ratio of acidic residues such as aspartic and glutamic acid as well as two phosphorylated residues. This protein is thought to be a calcium binding protein and to contribute to acceleration of bone mineralization.
Bone Sialoprotein is a highly glycosylated and sulphated phosphoprotein (MW=57,300). It has two stretches of polyglutamic acid which enable it to bind to hydroxyapatite. Since this protein contains RGD (arginine-glycine-aspartic acid), it has been suggested that it may mediate cell attachment. A number of potential sites for phosphorylation of serine, threonine and tyrosine, O- and N-glycosylation, and tyrosine sulfation are present in this molecule. BSP is expressed in differentiated osteoblasts at bone formation sites and is therefore, theorized to play an important role in bone formation (Shapiro et al., Matrix (1993)13(6):431).
Osteopontin (MW=44,000), is a phosphorylated glycoprotein (sialoprotein) having both - and O-glycosylation sites, which is found in mineralized bone matrix. It is composed of 301 amino acid residues and 13 of them are phosphorylated residues. Of the phosphorylated residues, 12 are phosphoserine (Pse) and 1 is phosphotyrosine (Pty). It has an RGD which is thought to be recognized by the cells. The exact function of this molecule has yet to be elucidated (Dodds et al., Journal of Bone and Mineral Research(1995) 10(11):1666-1680).
Proteoglycan in the bone is PG-I and PG-II and thought to control collagen formation.
In summary, bone matrix is composed of about 90% of Type I collagen and 10% of other minor functional proteins. These proteins (or glycoproteins) are mostly acidic as they contain a number of acidic amino acid residues (aspartic acid, glutamic acid, and gamma-carboxyl glutamic acid). In addition, a few glycosylated phosphoproteins containing number of phosphorylated amino acid residues have been isolated from bone matrix.
There have been a few theories developed regarding the potential role of endogenous phosphoproteins in the mineralization of type I collagen. However, as a relatively small fraction of those phosphoproteins are known to bound to collagenous matrix, the physiological role of the phosphoproteins has remained unclear.
In vitro studies have shown that phosphoproteins trapped in gelatin at very low concentrations promote initiation of the mineralization process (Boskey et al., Bone and Mineral (1990)11:55-65). When in solution, phosphoproteins enhance the conversion of amorphous calcium phosphate into hydroxyapatite (Nawrot et al., Biochemistry (1976) 51:3445-3449). At higher concentrations, however, phosphoproteins in solution inhibited the spontaneous precipitation or seeded growth of hydroxyapatite, which is thought to be an important step in matrix mineralization (Termine et al., Calcified Tissue Research (1976) 22:149-157; Udich et al., Biomed. Biochem. Acta. (1986)45:701-711; Doi et al., Arch. Oral Biol., (1992)37:15-21).
It has been shown that immobilized phosphoproteins may promote mineralization of the carrier material to which they are linked. Small amounts of dentinal phosphoproteins or egg yolk phosvitin covalently attached to Sepharose beads have been shown to induce mineral formation in vitro (Lussi et al., Arch Oral Biol (1988)33:685-691; Linde et al., Calcif. Tissue Int (1989)44:286-295). It was also shown that phosphate groups are important in enhancing the nucleation of a calcium-phosphorus solid phase by collagen-phosphoprotein complexes (Glimcher et al., Anat Rec (1989)224:139-153). In this study, however, the nature of the phosphoprotein-collagen complexes was poorly defined and little information was provided as to the precise experimental conditions that were used.
Another paper reported that phosphoproteins covalently bound to a collagenous matrix promote its mineralization in vitro. This report suggested that the rate of mineralization is influenced by both the amount and the nature of the bound organic hosphate groups. (Van Den Bos et al., Bone and Mineral (1993)23:81-93). In this study, the researchers used only differently phosphorylated rat dentin phosphoproteins as the potential accelerator of bovine bone mineralization and did not speculate as to any specific chemical structures which were responsible for such mineralization. Furthermore, the mineralization assays system used by this group included glycerophosphate which is a commonly used agent in ex vivo bone formation assays. Therefore, it is not clear whether the actual mineralization activity was caused by rat dentin phosphoproteins or glycerophosphate.
All of the theories pertaining to any correlation between collagen mineralization and phosphoproteins have been based upon in vitro experiments. No studies have been conducted using phosphoproteins or phosphopeptides in vivo animal models. This suggests that the current view in the art is that phosphoproteins affect bone mineralization through a specific local phenomenon in the bone.
Currently, there is no effective treatment for bone loss. Therapeutic agents such as estrogen, calcitonin, vitamin D, fluoride, Iprifravon, bisphosphonates, and a few others have failed to provide a satisfactory means of treatment. (Gennari et al., Drug Saf (1994) 11(3):179-95).
Estrogen and its analogues are frequently administered to patients with postmenopausal osteoporosis. Estrogen replacement therapy involves administration of estrogen just prior to or after the onset of menopause. However, as is often the case with steroid hornones, the long term use of estrogen has significant adverse effects such as breast and other gynecological cancers (Schneider et al., Int. J. Fertil. Menopausal Study (1995) 40(1):40-53).
Calcitonin, an endogenous hormone produced by the thyroid, binds selectively to osteoclasts, via its receptor, and inactivates them. Since the osteoclast is the only cell which can dissolve bone tissue, calcitonin binding can block or slow down bone degradation caused by the osteoclast. However, this biological mechanism is very short-lived, as the osteoclasts become tolerant to this drug relatively quickly. Therefore, the use of calcitonin does not provide an effective therapeutic option.
Fluoride has been shown to increase bone mass when it is administered to humans. However, while bone mass is increased, mechanical strength is not. Therefore, despite the increase in apparent bone mass, the risk of fracture remains (Fratzl et al., J. Bone Mineral Res. (1994)9(10):1541-1549). In addition, fluoride administration has significant health risks.
Iprifravon has been used to treat osteoporosis in limited areas in the world. However, the actual efficacy of this compound is questionable and it is not widely accepted as a useful therapeutic agent for bone diseases.
Bisphosphonates are compounds derivatized from pyrophosphate. Synthesis involves replacing an oxygen atom situated between two phosphorus atoms with carbon and modifying the carbon with various substituents. While bisphosphonates are known to suppress bone resorption, they have also been shown to inhibit bone formation. Furthermore, bisphosphonates adhere to the bone surface and remain there for very long time causing a long-term decrease in bone tissue turnover. As bone tissue needs to be turned over continuously, this decrease in turnover ultimately results in bone deterioration (Lufkin et al., Osteoporos. Int. (1994)4(6):320-322; Chapparel et al., J. Bone Miner. Res. (1995) 10(1):112-118).
Another significant problem with the agents described above is that with the exception of fluoride and iprifravon, they are unsuitable for oral administration, and thus, must be given parenterally. Since bone disorders are often chronic and require long-term therapy, it is important that therapeutic agents be suitable for oral administration.
In summary, a significant need exists for a therapeutic agent which can prevent or treat bone loss, or maintain or increase bone matrix, mineral content, and mechanical strength. A significant need also exists for an efficacious therapeutic agent which can be orally administered, is suitable for long-term use, and is free of significant adverse side-effects. The present inventors have satisfied this need by demonstrating in an in vivo osteoporosis model, that oral or parenteral administration of phosphopeptides significantly reduces bone loss and improves bone strength.